Vacuolar localization via the N-terminal domain of Sch9 is required for TORC1-dependent phosphorylation and downstream signal transduction

Abstract

The budding yeast Sch9 kinase (functional ortholog of the mammalian S6 kinase) is a major effector of the Target of Rapamycin Complex 1 (TORC1) complex in the regulation of cell growth in response to nutrient availability and stress. In budding yeast, Sch9 is partially localized at the vacuolar surface, where it is phosphorylated by TORC1 under favorable growth conditions. Sch9 recruitment at the vacuole is mediated by direct interaction between PI(3,5)P2 on the vacuolar membrane and the region of Sch9 encompassing amino acid residues 1-390, which contains a C2 domain. Since many C2 domains mediate phospholipid binding, it had been suggested that the C2 domain of Sch9 mediates its vacuolar recruitment. However, the in vivo requirement of the C2 domain for Sch9 localization had not been demonstrated, and the phenotypic consequences of Sch9 delocalization remained unknown. Here, by examining cellular localization, phosphorylation state and growth phenotypes of Sch9 truncation mutants, we show that deletion of the N-terminal domain of Sch9 (aa 1-182), but not the C2 domain (aa 183-399), impairs vacuolar localization and TORC1-dependent phosphorylation of Sch9, while causing growth defects similar those observed in sch9{Delta} cells. Artificial tethering of an N-terminally truncated Sch9 mutant at the vacuolar membrane rescued TORC1-dependent phosphorylation and cell growth. Our study uncovers a key role for the N-terminal domain of Sch9 and demonstrates that recruitment of Sch9 at the vacuolar surface is necessary for TORC1-dependent phosphorylation and downstream signal transduction for the regulation of cell growth.